Synthesis of steroid amino derivatives by recombinant transaminases and dehydrogenases

[N. Kaličanin]

122 str.

Hemijske nauke - Biohemija / Chemistry - Biochemistry

Upotreba enzima je važna za razvoj ekološki prihvatljivih i efikasnih procesa u sintezi hiralnih amina i amino steroida. Cilj ovog rada je bio dobijanje biokatalizatora pogodnog za sintezu amino derivata steroida, kao i drugih sekundarnih amina. Prvo je određena specifičnost transaminaze ArRMut11 (mutirane varijante enzima poreklom iz Arthrobacter sp.) ka različitim steroidima. Imobilizacijom na makroporoznom metakrilatnom polimeru dodatno je povećana stabilnost transaminaze ArRMut11. Imobilizovani enzim se mogao upotrebiti u više uzastopnih ciklusa sinteze 3α-amino-5α-androstan-17β-ola uz neznatan gubitak aktivnosti nakon 10 ciklusa upotrebe. Imobilizovani enzim je zadržao 75% početne aktivnosti nakon 2 h inkubacije u 35% dimetilformamidu na 70 °C, dok je aktivnost rastvornog enzima pri ovim uslovima pala na 19%. Razvoj biokatalizatora za sintezu sekundarnih amina obuhvatao je rekombinantu ekspresiju, prečišćavanje i karakterizaciju pretpostavljene opin dehidrogenaze iz halofilnog organizma Desulfohalobium retbaense u E. coli. Pokazano je da enzim predstavlja alanopin dehidrogenazu, da kao supstrate preferira L-alanin i piruvat, i druge manje voluminozne aminokiseline kao što su L-valin, L-serin i L-cistein. Enzim je pokazao izuzetnu stabilnost u opsegu pH od 6,0-11,0, u prisustvu NaCl do 3,0 M, kao i u 30% dimetil sulfoksidu. Navedene karakteristike čine ovaj enzim potencijalnim kandidatom za primenu u sintezi hiralnih sekundarnih amina i dalje unapređenje aktivnosti proteinskim inženjeringom. Enzim je podvrgnut nasumičnoj mutagenezi, i pretrazi biblioteka visokoefikasnom metodom zasnovanom na promeni fluorescencije NADH. Uočeno je nekoliko potencijalnih transformanata opin dehidrogenaze sa povećanom aktivnošću ili promenjenom specifičnošću u odnosu na divlji tip enzima.

Enzymes are important tools for the development of environmentally acceptable and efficient processes in the synthesis of chiral amines and amino steroids. The aim of this work was to obtain a biocatalyst suitable for the synthesis of steroid amino derivatives, as well as other secondary amines. First, the specificity of transaminase ArRMut11 (a mutated variant of the enzyme originating from Arthrobacter sp.) towards different steroids was determined. Immobilization on a macroporous methacrylate polymer additionally increased the stability of transaminase ArRMut11. The immobilized enzyme could be used in several consecutive cycles of 3α-amino-5α-androstan-17β-ol synthesis with a slight loss of activity after 10 cycles. The immobilized enzyme retained 75% of the initial activity after 2 h of incubation in 35% dimethylformamide at 70 °C, while the activity of the soluble enzyme under these conditions dropped to 19%. The development of a biocatalyst for the synthesis of secondary amines involved the recombinant expression, purification and characterization of a putative opine dehydrogenase from the halophilic organism Desulfohalobium retbaense in E. coli. It was shown that the enzyme is an allanopine dehydrogenase, that prefers L-alanine and pyruvate, and other less voluminous amino acids such as L-valine, L-serine and L-cysteine as substrates. The enzyme showed exceptional stability in the pH range of 6,0-11,0, in the presence of NaCl up to 3.0 M, as well as in 30% dimethyl sulfoxide. The mentioned characteristics make this enzyme a potential candidate for application in the synthesis of chiral secondary amines and further improvement of activity by protein engineering. The enzyme was subjected to random mutagenesis, and library screening using a high-throughput method based on NADH fluorescence change. Several potential opine dehydrogenase transformants with increased activity or altered specificity relative to the wild-type enzyme were observed.

hiralni amini, amino steroidi, transaminaze, opin dehidrogenaza, biokataliza, imobilizacija, halofili, metagenomika, proteinski inženjering

chiral amines, amino steroids, transaminases, opine dehydrogenase, biocatalysis, immobilization, halophiles, metagenomics, protein engineering

577.15:544.473(043.3)

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